The importance of a functional Krebs cycle for energy generation in the procyclic stage of Trypanosoma brucei was investigated under physiological conditions during logarithmic phase growth of a pleomorphic parasite strain. Wild type procyclic cells and mutants with targeted deletion of the gene coding for aconitase were derived by synchronous in vitro differentiation from wild type and mutant (⌬aco::NEO/⌬aco::HYG) bloodstream stage parasites, respectively, where aconitase is not expressed and is dispensable. No differences in intracellular levels of glycolytic and Krebs cycle intermediates were found in procyclic wild type and mutant cells, except for citrate that accumulated up to 90-fold in the mutants, confirming the absence of aconitase activity. Surprisingly, deletion of aconitase did not change differentiation nor the growth rate or the intracellular ATP/ADP ratio in those cells. Metabolic studies using radioactively labeled substrates and NMR analysis demonstrated that glucose and proline were not degraded via the Krebs cycle to CO 2 . Instead, glucose was degraded to acetate, succinate, and alanine, whereas proline was degraded to succinate. Importantly, there was absolutely no difference in the metabolic products released by wild type and aconitase knockout parasites, and both were for survival strictly dependent on respiration via the mitochondrial electron transport chain. Hence, although the Krebs cycle enzymes are present, procyclic T. brucei do not use Krebs cycle activity for energy generation, but the mitochondrial respiratory chain is essential for survival and growth. We therefore propose a revised model of the energy metabolism of procyclic T. brucei.Trypanosoma brucei, one of the causative agents of African trypanosomiasis, is a unicellular eukaryote, which during its life cycle alternates between the bloodstream of its mammalian host and the blood-feeding insect vector, the tsetse fly (Glossina spp.) (1). In the mammalian bloodstream, the long slender form of T. brucei proliferates, until at the peak of the parasitaemia nonproliferative short stumpy form cells accumulate that are prepared to differentiate to insect stage (procyclic form) parasites. In vivo this takes place in the tsetse midgut shortly after the insect blood meal. In culture this differentiation process (also referred to as transformation) can be induced by a temperature shift and the addition of millimolar concentrations of citrate or cis-aconitate (2), and in fully differentiation-competent trypanosome strains (termed pleomorphic) transformation is very rapid and perfectly synchronous (3-5).Differentiation of T. brucei is accompanied by several profound structural and biochemical changes, including the glucose metabolism. The long slender bloodstream form depends entirely on glycolysis for energy generation and excretes pyruvate as the major end product of carbohydrate metabolism (6 -8). In the procyclic stage, the end product of glycolysis, pyruvate, is not excreted but further metabolized inside the mitochondrion.

Background-Tetrahydrobiopterin (BH4), an essential cofactor for the synthesis of NO, improves endothelial dysfunction after ischemia/reperfusion. Therefore, we hypothesized that reduction of BH4 is involved in the attenuation of endothelium-dependent vasodilation in atherosclerosis, and we investigated the effect of alterations of the BH4 level on the vasodilatory potential of coronary resistance vessels from humans and pigs with atherosclerosis. Methods and Results-Coronary arterioles were obtained from patients undergoing CABG (atherosclerosis group) or valve replacement (control group) and from pigs fed either a standard diet (control group) or atherogenic diet (atherosclerosis group). After isolation, vessels were cannulated, pressurized, and placed on the stage of an inverted microscope. Dose-response curves were investigated in response to the endothelium-dependent agonists histamine, serotonin, and acetylcholine (for pigs, substance P) and to the endothelium-independent agonist sodium nitroprusside (SNP) under control conditions and before and after incubation of the vessels with sepiapterin (substrate for BH4 synthesis). In vessels from patients and from animals with atherosclerosis, compared with vessels from the control groups, there was a significant (PϽ0.05) reduction of vasodilation to all tested endothelium-dependent agonists but not to SNP. After application of sepiapterin, the responses to the endothelium-dependent agonists but not to SNP were significantly improved in vessels from the atherosclerosis groups. Sepiapterin did not influence vascular reactivity in the control groups. Conclusions-Atherosclerosis severely compromises endothelial function of coronary resistance arteries. Administration of sepiapterin leads to a significant improvement of endothelium-dependent vasodilatation to different agonists in vessels from humans and pigs with atherosclerosis. Therefore, we conclude that a reduced availability of BH4 is involved in the development of endothelial dysfunction in atherosclerosis.

In HHM, ubiquitin-related autophagic cell death and apoptosis cause a loss of myocytes. This plays an important role in progressive tissue damage and causes a reduction of the extent of functional recovery of HHM.

Formation of GTP by nucleoside diphosphate kinase (NDPK) can contribute to G protein activation in vitro.To study the effect of NDPK on G protein activity in living cells, the NDPK isoforms A and B were stably expressed in H10 cells, a cell line derived from neonatal rat cardiomyocytes. Overexpression of either NDPK isoform had no effect on cellular GTP and ATP levels, basal cAMP levels, basal adenylyl cyclase activity, and the expression of G s ␣ and G i ␣ proteins. However, co-expression of G s ␣ led to an increase in cAMP synthesis that was largely enhanced by the expression of NDPK B, but not NDPK A, and that was confirmed by direct measurement of adenylyl cyclase activity. Cells expressing an inactive NDPK B mutant (H118N) exhibited a decreased cAMP formation in response to G s ␣. Co-immunoprecipitation studies demonstrated a complex formation of the NDPK with G␤␥ dimers. The overexpression of NDPK B, but not its inactive mutant or NDPK A, increased the phosphorylation of G␤ subunits. In summary, our data demonstrate a specific NDPK B-mediated activation of a G protein in intact cells, which is apparently caused by formation of NDPK B⅐G␤␥ complexes and which appears to contribute to the receptor-independent activation of heterotrimeric G proteins. Nucleoside diphosphate kinase (NDPK)1 catalyzes the transfer of terminal phosphate groups from 5Ј-triphosphate to 5Ј-diphosphate nucleotides. In the cell, the major reaction is the phosphate transfer from ATP to other NDPs to maintain the levels of NTPs, especially the relatively high level of GTP. Only a small fraction of cellular NDPK binds to the plasma membrane, where it may serve the synthesis of GTP, required for the activation of G proteins (1-3). An activation of G proteins by NDPK has been disputed for more than 10 years. Although numerous in vitro studies (4 -7) have shown G protein activation through the enzymatic activity of NDPK (synthesis of GTP from a nucleoside triphosphate and GDP), the specificity of this phenomenon has been questioned (8, 9). Particularly in the intact cell, where GTP concentrations are in the upper micromolar range, evidence for a mechanism beyond the sole synthesis of GTP appears mandatory to support this hypothesis. On the other hand, we have shown recently (10) that NDPK activates G proteins and regulates adenylyl cyclase activity in canine cardiac sarcolemmal membranes. This activation required the catalytic activity of NDPK (synthesis of GTP) but was clearly distinct from the effect of exogenous GTP, suggesting a more direct interaction of NDPK and G proteins.Evaluation of direct G protein activation through phosphotransfer by NDPK is associated with substantial methodological constraints. Mainly, GDP is released spontaneously from G proteins and may then serve as a free substrate for phosphorylation by the NDPK (8). Approaches to immobilize the bound GDP at the G protein (11) are associated with protein denaturation, which in turn may lead to unspecific protein phosphorylation by the NDPK (12). In addition, structural con...

The accumulation and proliferation of vascular smooth muscle cells (VSMC) within the vessel wall is an important pathogenic feature in the development of atherosclerosis. Glucose metabolism has been implicated to play an important role in this cellular mechanism. To further elucidate the role of glucose metabolism in atherogenesis, glycolysis and its regulation have been investigated in proliferating VSMC. Platelet derived growth factor (PDGF BB)-induced proliferation of VSMCs significantly stimulated glucose flux through glycolysis. Further evaluating the enzymatic regulation of this pathway, the analysis of flux:metabolite co-responses revealed that anaerobic glycolytic flux is controlled at different sites of gycolysis in proliferating VSMCs, being consistent with the concept of multisite modulation. These findings indicate that regulation of glycolytic flux in proliferating VSMCs differs from traditional concepts of metabolic control of the Embden-Meyerhof pathway.